Triacylglycerol lipases (EC 3.1.1.3) are valued, efficient catalysts for a great variety of industrial uses, for example in the detergents industry, oil chemistry, the food industry and in the production of fine chemicals (Schmid R. D., Verger, R., Angew. Chem. Int. Ausg. 37: 1608-33 (1998)). Lipases are carboxylic ester hydrolases, which catalyze both the hydrolysis and the synthesis of triglycerides and other generally hydrophobic esters. All triacylglycerol lipases, whose three-dimensional crystal structure has been elucidated, belong to the α/β-hydrolase folding protein family, which have a similar overall architecture (Ollis D. L., Cheah, E., Cygler, M., Dijkstra, B., Frolow, F., Franken, S. M., Harel, M., Remingon, S. M., Silman, L., Schrag, J. D., Protein Eng. 5: 197-211 (1992)).
Candida antarctica-lipase B (also designated herein as CALB) is an efficient catalyst for many reactions and is used for example for stereoselective transformations and polyester synthesis (Anderson E. M., Larsson, K. M., Kirk, O., Biocat. Biotransform. 16: 181-204 (1998)) CALB has a solvent-accessible active center (Uppenberg J., Hansen, M. T., Patkar, S., Jones, A., Structure 2: 293-308 (1994)) and does not display interphase activation (Martinelle M., Holmquist M., Hult K., Biochim. Biophys. Acta 1258(3): 272-6 (1995)). The active center is a narrow funnel and for this reason CALB has a higher activity with respect to carboxylic acid esters, for example ethyl octanoate, than with respect to triglycerides (Martinelle 1995, supra). The fact that the activity of CALB in organic media is comparable to that in water, and in particular the high enantioselectivity of CALB for secondary alcohols make this enzyme one of the most important lipases currently in use in biotechnology.
Modification of CALB by random mutagenesis was described recently (Chodrorge M., Fourage L., Ullmann C., Duvivier V., Masson J. M., Lefèvre F., Adv. Synth. Catal. 347: 1022-1026 (2005). Several attempts to improve CALB for special applications through rational enzyme design have also been reported in the literature. Although some of these led to good results (Patkar S., Vind J., Kelstrup E., Christensen M. W., Svendsen A., Borch K., Kirk O., Chem. Phys. Lipids 93(1-2): 95-101 (1988); Rotticci D. “Understanding and Engineering the Enantioselectivity of Candida antarctica Lipase B towards sec-Alcohols”. Stockholm: Royal institute of Technology 1-61 (2000)), the possibilities for rational enzyme design are still limited through insufficient understanding of the catalytic properties of the enzyme.
Zhang et al. report in Protein Engineering, vol. 16, no. 8 (2003) 599 on experiments aiming at an improvement of the tolerance of CALB to irreversible thermal inactivation. By applying directed evolution techniques single mutants containing the mutation V210I, V221D or A281E were prepared. The double mutant (V210I, A281E) and the triple mutant (V210I, A281E, V221D) as well as the single mutant A281E showed a significant improvement of their melting point (Tm) versus the Tm of the wild-type enzyme.
Another approach for improving the activity and thermal stability of CALB was described by Suen et al. in Protein Engineering, Design & Selection, vol. 17, no. 2 (2004), 133. The technique of DNA family shuffling was used to create chimeric lipase B proteins derived from Candida antarctica and Crytococcus tzukubaensis as well as Hyphozyma sp. By high-throughput screening chimeras could be identified showing a higher activity towards the substrate 3-(3′,4′-dichlorophenyl)glutarate, an improved half-life at 45° C. and an improved melting point (Tm).
Magnusson et al. describe in J. Am. Chem. Soc. 123 (2001), 4354 mono-mutants of CALB which differ in their enantioselectivity with respect to the hydrolysis of the two ethylesters ethyl-3-hydroxybutanoat and ethyl-2-hydroxypropanoat. In particular, the mono-mutants T40A and T40V are described therein. The preparation of monoacylated polyols is not taught or suggested.
Rotticci et al. disclose in ChemBiochem. 2 (2001), 766 experiments for improving the enantioselectivity of CALB towards different optically active secondary mono-alcohols. In particular, the single mutants S47A, S47N, S47H, T42N, T42D, T42H, T42V, W104H, as well as the double mutant (T42V, S47A) have been prepared via rational design and further investigated.
Branneby et al. disclose in J. Am. Chem. Soc. 125 (2003), 874 the single mutants S105A and S105G of CALB and their behaviour during aldol condensation reactions.
Magnussen et al. disclose in ChemBiochem. (2005) 1051 mutants of CALB having an enlarged substrate pocket, which mutants have the ability to utilize larger secondary mono-alcohols. In particular, the single mutants T42V, S47A, W104A, W104H, W104Q and the double mutant (T42V, S47A) were investigated.
Consequently, none of the above-mentioned documents teaches or suggests making use of CALB enzymes in methods for monoacylating polyols, in particular non-cyclic polyols.
The selective preparation of monoacylated polyols is considered to be difficult to achieve because of the fact that a monoacylated intermediate is expected to be quickly further esterified by the same enzyme, so that monoesters of polyols are expected to be merely intermediary formed during the course of the reaction and with the progress of the esterification reaction the proportion of monoesterified products is more and more diminished.
Therefore, there was a continued need for providing an enzymatically catalyzed method for selectively monoacylating polyols, such as diols, in particular non-cyclic diols. In particular, there was a need fur methods allowing the improved, preferential preparation of monoacylated polyols. An improvement in this respect may be characterized by an increased maximum yield of monoester, an improved molar ratio of monoester product to higher or fully esterified products, as for example diesters, an improved molar ratio of monoester product to total esterified products, and/or a higher monoester yields at higher degrees of conversion.
There was a further need for methods for enantioselectively preparing such monoacylated polyols, in the case of enzyme substrates having an asymmetric carbon atom.